Researchers working to sequence mammalian-sized genomes today rely on nanopore-based methods that are hampered by two significant bottlenecks that limit precision and hinder progress in the field, says theoretical physicist Murugappan Muthukumar of polymer science and engineering. He has a four-year, $1.39 million grant from NIH’s National Human Genome Research Institute to overcome these hurdles to make way for a new wave of low-cost, rapid, high-quality sequencing technologies.
“Right now, DNA reading accuracy in the nanopore-based method is about 95 percent,” he points out. “We want to improve that.”
With the current method of sequencing DNA, Muthukumar explains, nucleic acid units in a DNA strand pass through a 1.5-nanometer opening, or pore, one at a time where they are read and recorded, “like unwinding a ball of wool and passing it through the eye of a needle,” he says. Except that in the case of DNA waiting to be read, it is not neatly coiled like a ball of yarn, but is jumbled, disordered and influenced by electrical and entropic forces, water currents, temperature and salt ions.
It’s already a fast process, but the system includes considerable thermal “noise” at the interrogation point, which introduces reading errors. In addition, such systems face difficulties at controlling and capturing large DNA molecules at the mouth of the nanopore. It’s particularly difficult to locate the strand beginning to start the process, the researcher says.
For tackling these two critical challenges, Muthukumar and his team including postdoctoral researchers Ining “Amy” Jou, Zachary Dell and graduate research assistants Sadhana Chalise andKhatcher Margossian, propose to build a “computational design engine” to enable sequencing of DNA and RNA at the maximum accuracy allowed by the laws of physics.
As Muthukumar explains, “This is a polymer physics problem because the thread-like DNA can adopt many conformations – coiled, swirled, not straight – yet you have to navigate the polymer thread toward the nanopore. For this, it helps to enumerate the number of allowable conformations and evaluate the probable locations of the ends of the DNA molecule. Then it is necessary to navigate one end of a strand of DNA, which is buried in this morass, and bring it to the entrance of the nanopore and thread it, one base at a time.”
He adds, “Navigating one of the two ends of a long DNA strand to the pore is a very daunting task and our goal is to find strategies to facilitate the capture of the chain end at the pore, using water flow and electrical forces.” Here, his team will attempt to answer the question, “What is the maximum size of DNA that can be sequenced using this technology within the specified time limit for sequencing an individual’s genome,” because knowing the statistical probability of accurate sequencing success for various sample sizes is one factor that will help researchers who want to sequence large-sized DNA in this manner.
“Right now the technologists can only sequence 1,000 or 10,000 base pairs in typical single molecule electrophoresis experiments, but we want to capture much longer DNA strands,” he notes. “It’s a very challenging problem, but we do know how to compute the conformational probabilities of finding an end and how long it will take for that end to unravel enough to get to the target. We hope to be able to say, given 10 minutes or a certain fixed time of sequencing, how big a DNA you can capture and sequence while still retaining high accuracy.”
“One of the goals is to reduce the randomness of motion to make it steady. We have derived a theorem where there is a limit for accurate positioning of the nucleotide at the interrogation point,” Muthukumar adds.
As for the “noise” problem at the nanopore interrogation point, Muthukumar’s team plans to model the application of an alternating current (AC) electric field at different frequencies to arrive at the optimal regulation of speed to reduce thermal noise as sequences are read. “We want to design technical strategies for regulating the speed,” he notes. “We’ll try to accomplish that by adding an external oscillatory field.”
“The challenge is that when I bring the polymer through the hole, it doesn't move in a regular fashion,” he explains. “The DNA is pushed and pulled, two steps forward one step back, which makes reading inaccurate. By applying an AC field, we can elicit stochastic resonance to synchronize the movement of the DNA with the externally applied force, by redistributing energy from the thermal noise. With this technique, the reading device does not change, but now it can be more accurate because it forces the DNA to march forward in a predictable rhythm.”
“This phenomenon of movement of large macromolecules through a nanopore is a ubiquitous problem in biology and many health care technologies,” the researcher points out. “This work has a fundamental value. This is also technology needed by many DNA-reading companies who need to read sequences correctly for disease diagnostics. There is a race in this area now. The goal is to understand how this phenomenon takes place and how to transfer our understanding to design.”
“Our strategies are designed for DNA sequencing, but the findings will have many uses for other contexts in biology. This could be used for RNA sequencing, which is even more complex,”Muthukumar says.